Fig. 3
Effect of mutations in HaRxL103 alleles on in planta subcellular localization and recognition by RPP4. a Schematic structures of HaRxL103 alleles. Filled and diagonal boxes indicate an N-terminal signal peptide and an RxLR motif, respectively. Red-letter residues indicate polymorphic sites. E, Glu; H, His; R, Arg; D, Asp; K, Lys; Q, Gln. b Subcellular localization of HaRxL103 alleles. GFP-tagged HaRxL103 alleles were transiently (co)expressed with/without RPP4-FLAG via agroinfiltration in N. benthamiana. Images are from GFP channel and single-plane confocal images. Scale bars, 10 μm. c HR cell death phenotypes when co-expressed of HaRxL103 alleles with RPP4 in N. benthamiana. The leaves inoculated with Agrobacterium containing the indicated gene constructs were photographed under UV at 3 dpi. Protein accumulation (d), subcellular localization (e), AtPR1 expression (f), and Hpa growth (g) in Arabidopsis Col-0 transgenic lines containing Est-GFP, Est-103Emoy2 and estradiol-inducible GFP-HaRxL103Hind2 (Est-103Hind2) constructs. Immunoblot, qRT-PCR and Hpa growth analyses were done as described in Fig. 2d–f. d Asterisks indicate the detected GFP, GFP-HaRxL103Emoy2 or GFP-HaRxL103Hind2 constructs. e Col-0 transgenic lines pretreated with estradiol were DAPI-stained. The upper image is from the GFP channel, the middle image is from the DAPI channel, and the lower image is the overlay of the GFP and DAPI channels. Scale bars, 10 μm. f Data are means ± SDs from three biological replicates. Different letters indicate significantly different values at p < 0.01 (one-way ANOVA, Tukey’s HSD). g Data are means ± SEs from five biological replicates. Different letters indicate significantly different values at p < 0.01 (one-way ANOVA, Tukey’s HSD)
